Investigators have recently turned their attention to understanding the biological mechanisms of taste, and in particular bitter taste. For a review of the literature see, for example, Caicedo A. and Roper S D. (2001) Science 291: 1557-1560; Dulac C. (2000) Cell 100: 607-610; Kinnamon S. C. (2000) Neuron 25: 507-510; Lindemann B. (2001) Nature 413: 219-225.; and Margolskee R F. (2001) J. Biol. Chem. 277: 1-4.
Bitter taste is aversive, and as such provides humans with a mechanism of protection against poisonous substances, which are generally bitter-tasting compounds. More subtly, bitter-tastants also affect the palatability of food, beverages, thereby influencing human nutritional habits as is more fully discussed by Drewnowski in “The Science and Complexity of Bitter Taste”, (2001) Nutr. Rev. 59: 163-169. They also affect the palatability of other ingestibles such as orally administered pharmaceuticals and nutraceuticals. Understanding the mechanism of bitter taste transduction has implications for the food and pharmaceutical industries. If the bitter taste transduction pathway can be manipulated, it may be possible to suppress or eliminate bitter taste to render foods more palatable and increase patient compliance with oral pharmaceutics.
Taste transduction involves the interaction of molecules, i.e. tastants with taste receptor-expressing cells which reside in the taste buds located in the papillae of the tongue. Taste buds relay information to the brain on the nutrient content of food and the presence of poisons. Recent advances in biochemical and physiological studies have enabled researchers to conclude that bitter taste transduction is mediated by so-called G-protein coupled receptors (GPCRs). GPCRs are 7 transmembrane domain cell surface proteins that amplify signals generated at a cell surface when the receptor interacts with a ligand (a tastant) whereupon they activate heterotrimeric G-proteins. The G-proteins are protein complexes that are composed of alpha and beta-gamma sub-units. They are usually referred to by their alpha subunits and classified generally into 4 groups: G alpha s, i, q and 12. The G alpha q type couple with GPCRs to activate phospholipase C which leads to an increase in cellular Ca2+. There are many Gq-type G-proteins that are promiscuous and can couple to GPCRs, including taste receptors, and these so-called “promiscuous” G-proteins are well known in the art. These G-proteins dissociate into alpha and beta-gamma subunits upon activation, resulting in a complex cascade of cellular events that result in the cell producing second messengers, such as calcium ions, that enable the cells to send a signal to the brain indicating a bitter response.
There is also anatomical evidence that GPCRs mediate bitter taste transduction: clusters of these receptors are found in mammalian taste cells containing gustducin. Gustducin is a G-protein subunit that is implicated in the perception of bitter taste in mammals see, for example, Chandrashekar, J. et al. (2000) Cell 100: 703-711; Matsunami H. et al. (2000) Nature 404: 601-604; or Adler E. et al. (2000) Cell 100: 693-702. cDNAs encoding such GPCRs have been identified, isolated, and used as templates to compare with DNA libraries using in-silico data-mining techniques to identify other related receptors. In this manner it has been possible to identify a family of related receptors, the so-called T2R or TAS2R family of receptors, which have been putatively assigned as bitter taste receptors.
Humans are able to detect with a limited genetic repertoire of about 30 receptor genes thousands of different bitter compounds. Since their discovery in the year 2000 (Adler E. et al. (2000) supra; Chandrashekar J. et al. (2000) supra; Matsunami H. et al (2000) supra) only few mammalian TAS2R5 have been deorphanised, i.e. ligands, in particular agonists have been identified. The murine mTAS2R5 (Chandrashekar J. et al (2000) supra) and the rat rTAS2R9 (Bufe B. et al. (2002) Nature Genetics 32:397-401) respond to the toxic bitter substance cycloheximide, the mouse mTAS2R8 and the human hTAS2R4 respond to high doses of denatonium and, to a lesser extent, to 6-n-propyl-2-thiouracil (Chandrashekar J. et al. (2000) supra), the human hTAS2R10 and hTAS2R16 respond selectively to strychnine and bitter glucopyranosides, respectively (Bufe B. et al (2002) supra). Human hTAS2R46 was characterized as a bitter receptor broadly tuned to sesquiterpene lactones and to clerodane and labdane diterpenoids as well as to the unrelated bitter substances strychnine and denatonium (Brockhoff A. et al., (2007) J. Agric. Food Chem. 55(15): 6236-6243). Although for some TAS2Rs a limited promiscuity (mTAS2R8, hTAS2R4) or specificity for a group of chemically related compounds (hTAS2R16) was reported, the relative selectivity of ligand recognition by the receptors published to date does by far not explain the enormous number of bitter tastants recognised by the mammalian gustatory system. Also very little is known about substances that can act as antagonists of bitter taste receptors and thereby reduce or suppress a bitter taste sensation.
The knowledge about compounds that act as bitter receptor antagonists is prerequisite to the implementation of a method to isolate structurally related antagonists which may be at least as potent in suppressing the bitter taste receptor activity as the original antagonist and which may feature additional advantages such as lower toxicity, better solubility, improved stability and so forth. A bitter taste receptor antagonist isolated by such method can also be isolated and modified or combined with other bitter taste receptor antagonists in such a way that it is capable of targeting a broader range of known bitter taste receptors with high affinity to achieve a more effective suppression of bitter taste.
The present invention provides compounds which act as antagonists for the function of human bitter taste receptors hTAS2R40, hTAS2R43, hTAS2R44, hTAS2R46, and hTAS2R47. The disclosure of the present patent application allows the implementation of a method to isolate additional structurally related antagonists or agonists for these six bitter taste receptors to suppress or to enhance, respectively, bitter taste. Furthermore, the use of such antagonists and agonists is disclosed.